The present invention relates to a novel, reduced beta-nicotinamide adenine dinucleotide-dependent carbonyl reductase that is useful for producing alcohol, particularly (S)-4-halo-3-hydroxybutyrate ester; DNA encoding said enzyme; a method for producing said enzyme; and a method for producing alcohol, particularly (S)-4-halo-3-hydroxybutyrate ester, using said enzyme.
Asymmetric reduction methods using microorganisms such as baker""s yeast to produce optically active (S)-4-halo-3-hydroxybutyrate ester (Unexamined Published Japanese Patent Application No. (JP-A) Sho 61-146191, JP-A Hei 6-209782, and so on) have been known for some time. These production methods, however, have problems that must be solved for industrial applications because the optical purity and yield of the product are low due to more than one reductases existing in microbial cells. Optically active (S)-4-halo-3-hydroxybutyrate ester is utilized as a synthetic intermediate of drugs. It is thus important in the chemical industry to determine how to produce (synthesize or resolve) optically purified antipodes of the compound.
Enzymes capable of producing (S)-4-halo-3-hydroxybutyrate ester from 4-haloacetoacetate ester are described below, and methods for synthesizing (S)-4-halo-3-hydroxybutyrate ester using these enzymes have been reported.
3-Hydroxysteroid dehydrogenase (JP-A Hei 1-277494)
Glycerol dehydrogenase (Tetrahedron Lett. 29, 2453-2454 (1988))
Alcohol dehydrogenase derived from Pseudomonas sp. PED (J. Org. Chem. 57, 1526-1532 (1992))
Reductases derived from baker""s yeast (D-enzyme-1, D-enzyme-2, J. Am. Chem. Soc. 107, 2993-2994 (1985))
Aldehyde dehydrogenase 2 derived from Sporobolomyces salmonicolor (Abstract of 391st Meeting of the Kansai Branch of the Japan Society of Bioscience, Biotechnology, and Agrochemistry, p37 (1995))
Ketopantothenate reductase derived from Candida macedoniensis (Arch. Biochem. Biophys. 294, 469-474 (1992))
Ethyl 4-chloroacetoacetate reductase derived from Geotrichum candidum (Enzyme Microb. Technol. 14, 731-738 (1992))
Carbonyl reductase derived from Candida magnoliae (W098/35025)
Carbonyl reductase derived from Kluyveromyces lactis (JP-A Hei 11-187869)
Most of these enzymes are reductases that require reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme. Thus, the method for synthesizing (S)-4-halo-3-hydroxybutyrate ester using these enzymes is industrially disadvantageous because it needs the addition and regeneration of expensive and chemically unstable NADPH.
3-Hydroxysteroid dehydrogenase, glycerol dehydrogenase, and alcohol dehydrogenase derived from Pseudomonas sp. PED are oxidoreductases, which catalyze not only reduction reactions using reduced nicotinamide adenine dinucleotide (NADH) as an electron donor but also oxidation (dehydrogenation) reactions. The use of these enzymes cannot produce (S)-4-halo-3-hydroxybutyrate ester in a high yield because the equilibrium of the enzymatic reaction is likely to limit the reaction rate.
An objective of this invention is to provide a carbonyl reductase that uses NADH as a coenzyme. Another objective of this invention is to provide a carbonyl reductase with excellent stereoselectivity capable of acting on a substrate, 4-haloacetoaectate ester, to produce optically active (S)-4-halo-3-hydroxybutyrate ester with high optical purity at a high yield.
Still another objective of the present invention is to isolate a DNA encoding the carbonyl reductase with desired properties and obtaining a recombinant enzyme. A further objective of the invention is to provide a method for enzymatically producing optically active (S)-4-halo-3-hydroxybutyrate ester using a novel carbonyl reductase.
Another objective of the invention is to obtain a recombinant capable of simultaneously expressing not only the desired enzyme described above but also an enzyme that reduces NAD+ to NADH. It is also an objective of the invention to provide a method for enzymatically producing optically active (S)-4-halo-3-hydroxybutyrate ester using a novel carbonyl reductase, involving an enhanced regeneration system of the coenzyme using the above recombinant.
The present inventors thought that carbonyl reductases capable of utilizing NADH as an electron donor would be industrially useful since NADH is less expensive and chemically more stable than NADPH. We also thought that enzymes that reduce 4-haloacetoacetate ester to form (S)-4-halo-3-hydroxybutyrate ester but do not substantially dehydrogenate the formed (S)-4-halo-3-hydroxybutyrate ester would be useful for efficiently producing optically active (S)-4-halo-3-hydroxybutyrate ester.
The present inventors sought enzymes that meet the above requirements and found a desired enzyme from Kluyveromyces aestuarii. 
We succeeded in isolating a novel enzyme and DNA encoding the enzyme, and in developing a method for producing alcohol using this enzyme. The present invention relates to the carbonyl reductase described below, DNA encoding said enzyme, a method for producing said enzyme, and the use of said enzyme.
1. A carbonyl reductase having the following physicochemical properties: Reactivity
It reduces 4-haloacetoacetate ester to produce (S)-4-halo-3 hydroxybutyrate ester using reduced beta-nicotinamide adenine dinucleotide as an electron donor.
Substrate specificity
It has high reductase activity for 4-chloroacetoacetate ester but does not substantially dehydrogenate any optical isomers of 4-halo-3-hydroxybutyrate ester and
shows higher enzymatic activity when used with reduced beta-nicotinamide adenine dinucleotide as an electron donor than reduced beta-nicotinamide adenine dinucleotide phosphate.
2. The carbonyl reductase described in 1, which has additional physicochemical properties below:
Optimal pH
5.0to 6.0
Substrate specificity
It does not substantially dehydrogenate isopropanol and does not reduce acetoacetate. Molecular weight
About 32,000 when determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis.
3. A substantially pure polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 and having the enzymatic activity for catalyzing the reduction of 4-haloacetoacetate ester to (S)-4-halo-3-hydroxybutyrate ester using reduced beta-nicotinamide adenine dinucleotide as an electron donor.
4. A substantially pure polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 containing up to 30 conservative amino acid substitutions, and having the following enzymatic activities:
reduces 4-haloacetoacetate ester to produce (S)-4-halo-3-hydroxybutyrate ester using reduced beta-nicotinamide adenine dinucleotide as an electron donor;
has high reductase activity for 4-chloroacetoacetate ester but does not substantially dehydrogenate any optical isomers of 4-halo-3-hydroxy-butyrate ester; and
shows higher enzymatic activity when used with reduced beta-nicotinamide adenine dinucleotide as an electron donor than reduced beta-nicotinamide adenine dinucleotide phosphate.
5. A substantially pure polypeptide encoded by a nucleic acid that hybridizes with the nucleic acid consisting the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, and having the following enzymatic activities:
reduces 4-haloacetoacetate ester to produce (S)-4-halo-3-hydroxybutyrate ester using reduced beta-nicotinamide adenine dinucleotide as an electron donor;
has high reductase activity for 4-chloroacetoacetate ester but does not substantially dehydrogenate any optical isomers of 4-halo-3-hydroxybutyrate ester; and
shows higher enzymatic activity when used with reduced beta-nicotinamide adenine dinucleotide as an electron donor than reduced beta-nicotinamide adenine dinucleotide phosphate.
6. The substantially pure polypeptide described in 5, comprising an amino acid sequence having at least 70% homology with the amino acid sequence represented by SEQ ID NO: 2.
7. An isolated nucleic acid encoding the polypeptide described in 3.
8. An isolated nucleic acid encoding the polypeptide described in 4.
9. An isolated nucleic acid encoding the polypeptide described in 5.
10. An isolated nucleic acid encoding the polypeptide described in 3 comprising the nucleotide sequence represented by SEQ ID NO: 1.
11. An isolated nucleic acid hybridizing with the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, and encoding a polypeptide having the following enzymatic activities:
reduces 4-haloacetoacetate ester to produce (S)-4-halo-3-hydroxybutyrate ester using reduced beta-nicotinamide adenine dinucleotide as an electron donor;
has high reductase activity for 4-chloroacetoacetate ester but does not substantially dehydrogenate any optical isomers of 4-halo-3-hydroxybutyrate ester; and
shows higher enzymatic activity when used with reduced beta-nicotinamide adenine dinucleotide as an electron donor than reduced beta-nicotinamide adenine dinucleotide phosphate.
12. The nucleic acid of claim 11 comprising a nucleotide sequence having at least 70% homology with the nucleotide sequence represented by SEQ ID NO: 1.
13. A recombinant vector comprising the nucleic acid described in 7.
14. A recombinant vector comprising the nucleic acid described in 8.
15. A recombinant vector comprising the nucleic acid described in 9.
16. A transformant carrying the vector described in 13.
17. A transformant carrying the vector described in 14.
18. A transformant carrying the vector described in 15.
19. The transformant described in 16, which is a microorganism.
20. A method for producing a carbonyl reductase, the method comprising culturing the transformant described in 16.
21. A recombinant vector comprising the nucleic acid described in 7 and the nucleic acid encoding a glucose dehydrogenase.
22. The vector described in 21, wherein a glucose dehydrogenase is derived from Bacillus subtilis. 
23. A transformant carrying the vector described in 21.
24. The transformant described in 21, which is a microorganism.
25. A method for producing the enzyme described in 1, the method comprising culturing a microorganism belonging to the genus Kluyveromyces and producing the enzyme described in 1.
26. The method for producing the enzyme described in 25, wherein the enzyme comprises the amino acid sequence represented by SEQ ID NO: 2.
27. The method for producing the enzyme described in 25, wherein the microorganism belonging to the genus Kluyveromyces is Kluyveromyces aestuarii. 
28. A method for producing a polypeptide encoded by the nucleic acid described in 7, the method comprising culturing the transformant described in 16.
29. The method for producing the polypeptide described in 28, wherein the transformant is a microorganism.
30. A method for producing alcohol, the method comprising reacting ketone with the carbonyl reductase described in 1, microorganisms producing it, or treated microorganisms.
31. The method for producing alcohol, wherein the carbonyl reductase comprises the amino acid sequence represented by SEQ ID NO: 2.
32. The method for producing alcohol described in 30, wherein the microorganism is the transformant described in 16.
33. The method for producing alcohol described in 30, wherein ketone is a derivative of 4-haloactoacetate ester, and alcohol is a derivative of (S)-4-halo-3-hydroxybutyrate ester.
34. The method for producing alcohol described in 33, wherein the derivative of ethyl 4-haloactoacetate is 4-chloroacetoacetate ester, and alcohol is ethyl (S)-4-chloro-3-hydroxybutyrate.
35. The method for producing alcohol described in 30, the method further comprising converting oxidized beta-nicotinamide adenine dinucleotide to its reduced form.
36. The method for producing alcohol described in 35, wherein oxidized beta-nicotinamide adenine dinucleotide is reduced by a conversion of glucose to delta-gluconolactone by using a glucose dehydrogenase.
37. The method for producing alcohol described in 36, wherein glucose dehydrogenase is expressed by the transformant described in 23.